CN111996243A - Method for rapidly detecting EGFRvIII mutation - Google Patents
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Abstract
The invention discloses a method for rapidly detecting EGFRvIII mutation. The EGFRvIII RNA is used as an initial template of the reaction, a lengthy intron sequence in an EGFRvIII DNA sequence is avoided, and the probe spans the fusion site of the EGFRvIII exon, so that the interference of genome DNA is effectively avoided, and the detection result is more accurate; the reverse transcription and the real-time PCR share a reaction system, and compared with the traditional reverse transcription, the detection is carried out after the amplification by taking cDNA as a template, so that the pollution possibly caused by multiple uncapping is avoided; the housekeeping gene ACTB is introduced into a single system through the change of the fluorescent group, so that the risk of false negative is reduced, and the result is interpreted through the delta Ct, so that the risk of false positive is reduced; the designed PCR product is relatively small, so that the paraffin-embedded sample with high degradation degree can also meet the detection requirement, and meanwhile, the detection result can correspond to the EGFRvIII immunohistochemical result of the same tissue sample. The method is simple in operation, and reliability and accuracy of the detection result are guaranteed.
Description
Technical Field
The invention relates to the field of cancer detection and molecular biology, in particular to a method for rapidly detecting EGFRvIII mutation.
Background
Malignant tumor is one of the diseases which seriously affect human health at present, and the internal cause of the malignant tumor is that the cell growth regulation is abnormal due to the change of genes in cells, so that abnormal hyperplasia is caused, and then the tumor is generated. Numerous studies have shown that egfr (epidermal Growth Factor receptor) protein, one of the surface Growth Factor receptor family members, is involved in the inhibition of the regulatory mechanisms of tumor cell proliferation, angiogenesis, tumor invasion, metastasis, apoptosis, and the like. Through the combination with ligand, EGFR receptor can activate downstream signal channel, so as to regulate and control the physiological processes of cell growth, proliferation, differentiation and the like, therefore, the EGFR signal channel is over-activated, which can lead to the uncontrolled cell growth.
EGFRvIII is a common mutant of EGFR, is closely related to various malignant phenotypes of human brain glioma, and is caused by EGFRvIII in up to 30 percent of malignant cerebroma. Compared with wild EGFR, EGFRvIII lacks extracellular exons 2-7, namely ligand binding region, so that the EGFRvIII can activate a signal path without being bound with a ligand, and then cell growth is out of control. The EGFRvIII is only expressed in tumor cells and is one of important targets for targeted drug research in recent years, and the rapid and accurate detection of the expression condition of the EGFRvIII in tumor tissues is the key of targeted therapy. Immunohistochemistry or Western Blot is an important way to detect protein level expression of EGFRvIII, however, numerous studies have shown that the association between EGFRvIII protein level expression and nucleic acid level is weak. Therefore, in the detection of EGFRvIII, it is usually necessary to consider both protein and nucleic acid levels.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention provides a method for rapidly detecting EGFRvIII mutation.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for rapidly detecting EGFRvIII mutation comprises the following steps:
(1) extracting RNA of the tumor tissue;
(2) constructing a PCR reaction system;
(3) performing real-time fluorescent quantitative PCR by a TaqMan probe method;
(4) and (5) judging the result.
The method for rapidly detecting the EGFRvIII mutation comprises the following steps of (2): adding reverse transcriptase, polymerase, EGFRvIII upstream and downstream primers, an EGFRvIII TaqMan probe, an ACTB upstream and downstream primer and an ACTB TaqMan probe into the same reaction system by taking the RNA extracted in the step (1) as a template; the three processes of cDNA construction, PCR product amplification and fluorescent signal collection are realized by changing the reaction temperature of a PCR reaction system.
The method for rapidly detecting the EGFRvIII mutation comprises the following steps of (4): determining whether the sample is positive for the EGFRvIII mutation or not according to the numerical size of the delta Ct, wherein the delta Ct is Ct (EGFRvIII) -Ct (ACTB); if delta Ct is more than 0 and less than 10, the sample is positive for EGFRvIII mutation; increasing the dosage of RNA and detecting again, wherein the dosage of the delta Ct is 10; and the delta Ct is more than 10 or no EGFRvIII specific fluorescent new number is collected, and the sample is EGFRvIII negative.
In the method for rapidly detecting the EGFRvIII mutation, the sequence of the EGFRvIII upstream primer is 5 'AGTCGGGCTCTGGAGGAAA-3', the sequence of the EGFRvIII downstream primer is 5 'TCCATCTCATAGCTGTCGGC-3', and the sequence of the EGFRvIII TaqMan probe is 5 '-FAM-AGAAAGGTAATTAT-MGB-3'; the sequence of the ACTB upstream primer is 5 'CTTCGCGGGCGACGAT-3', the sequence of the ACTB downstream primer is 5 'TAGGAATCCTTCTGACCCATGC-3', and the sequence of the ACTB TaqMan probe is 5 '-HEX-CGGGCCGTCTTCCC-MGB-3'.
The invention has the beneficial effects that: (1) according to the invention, the EGFRvIII RNA is used as an initial template of reaction, a lengthy intron sequence in an EGFRvIII DNA sequence is avoided, and a probe spans a fusion site of the EGFRvIII exon, so that the interference of genome DNA is effectively avoided, and the detection result is more accurate; (2) in the invention, reverse transcription and real-time PCR share a reaction system, cDNA construction with RNA as a template, PCR amplification with cDNA as a template and detection of products by a Taqman probe are completed, compared with the traditional detection method which adopts cDNA as a template after reverse transcription and then detection, the steps are more simplified, a single system is constructed, and the pollution possibly caused by multiple uncapping is avoided; (3) the method adopts Taqman real-time PCR to detect the mutation condition of the EGFRvIII, avoids possible pollution caused by uncovering compared with agarose gel electrophoresis, and is more sensitive and accurate than an SYBR Green real-time PCR method by using a Taqman specific probe; (4) in the invention, the housekeeping gene ACTB is introduced into a single system through the change of the fluorescent group, so that the existence of negative control is ensured, and the risk of false negative is reduced; the result is interpreted by the delta Ct, so that the risk of false positive is reduced, and the experimental result is more accurate and credible; (5) the PCR product designed by the invention is relatively small, so that the paraffin-embedded sample with higher degradation degree can also meet the detection requirement, and meanwhile, the detection result can correspond to the EGFRvIII immunohistochemical result of the same tissue sample. In conclusion, the detection method is simple to operate, redundant steps and possible pollution are effectively avoided, and the reliability and accuracy of the detection result are ensured by adding contrast while the detection is simple.
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The invention is further illustrated with reference to the following figures and examples.
FIG. 1 is a TaqMan real-time PCR amplification profile of sample 1 in example 1;
FIG. 2 is a sample 2TaqMan real-time PCR amplification profile in example 2;
FIG. 3 shows immunohistochemical results for sample 1 in example 1;
FIG. 4 shows the results of electrophoresis of sample 1 and sample 2;
FIG. 5 shows the sequencing results of sample 1 and sample 2.
Detailed Description
In order to make the technical solutions of the present invention better understood, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
[ example 1 ]
A method for rapidly detecting EGFRvIII mutation comprises the following steps:
(1) extracting RNA of tumor tissue of the sample 1;
(2) constructing a PCR reaction system;
(3) performing real-time fluorescent quantitative PCR by a TaqMan probe method;
(4) and (5) judging the result.
Further, the extraction procedure for sample 1RNA was as follows: dewaxing a paraffin sample: 5 paraffin tissue sections of 7 μm samples 1 were placed in a 1.5ml centrifuge tube, 1ml xylene was added, centrifugation was carried out at 13,000Xg for 1 minute, the supernatant was discarded, 1ml xylene was added, centrifugation was carried out at 13,000Xg for 1 minute, and the supernatant was discarded. Adding 1ml of absolute ethyl alcohol into a centrifuge tube, centrifuging for 1 minute at 13,000Xg, removing supernatant, and airing; adding tissue lysate and proteinase K into the air-dried tissue, splitting at 56 ℃ for 15 minutes, transferring the centrifuge tube to 80 ℃, keeping for 15 minutes, taking down, adding DNase into the centrifuge tube after the centrifuge tube is moved to room temperature, and standing for 15 minutes at room temperature; adding a binding buffer solution and absolute ethyl alcohol into a sample, gently mixing uniformly, transferring the sample to an RNA extraction filter column, centrifuging for 1 minute at 13,000Xg, adding 600 mu L of a washing buffer solution into the sample, centrifuging for 1 minute at 13,000Xg, and repeatedly washing for three times; putting the filter column into a new 1.5ml centrifuge tube, placing the centrifuge tube at 56 ℃, opening the cover, airing for 3 minutes, adding eluent, covering the cover, and keeping at 56 ℃ for 2 minutes; the sample 1RNA sample was obtained by centrifugation at 13,000Xg for 1 minute.
Further, the PCR reaction system is constructed as follows: firstly, 2 mu L of RNA is taken as a template for constructing cDNA, and a housekeeping gene ACTB is introduced into a reaction system, so that the existence of negative control is ensured, the risk of false positive is reduced, the result interpretation is facilitated, and the specific reaction system comprises the following components:
the three processes of cDNA construction, PCR product amplification and fluorescent signal collection are realized by changing the reaction temperature of a reaction system, and the specific reaction program is as follows:
and after the reaction system is constructed, performing TaqMan real-time PCR to detect the EGFRvIII mutation condition.
Further, the step (4) is specifically as follows: determining whether the sample is positive for the EGFRvIII mutation or not according to the numerical size of the delta Ct, wherein the delta Ct is Ct (EGFRvIII) -Ct (ACTB); if delta Ct is more than 0 and less than 10, the sample is positive for EGFRvIII mutation; increasing the dosage of RNA and detecting again, wherein the dosage of the delta Ct is 10; and the delta Ct is more than 10 or no EGFRvIII specific fluorescent new number is collected, and the sample is EGFRvIII negative.
Further, the sequence of the EGFRvIII upstream primer is 5 'AGTCGGGCTCTGGAGGAAA-3', the sequence of the EGFRvIII downstream primer is 5 'TCCATCTCATAGCTGTCGGC-3', and the sequence of the EGFRvIII TaqMan probe is 5 '-FAM-AGAAAGGTAATTAT-MGB-3'; the sequence of the ACTB upstream primer is 5 'CTTCGCGGGCGACGAT-3', the sequence of the ACTB downstream primer is 5 'ATAGGAATCCTTCTGACCCATGC-3', and the sequence of the ACTB TaqMan probe is 5 '-HEX-CGGGCCGTCTTCCC-MGB-3'.
The amplification map obtained by the sample 1TaqMan real-time PCR of the present example is shown in FIG. 1: ● in FIG. 1 represents the HEX fluorescence signal, the negative control ACTB; ■ represents the FAM fluorescence signal, i.e. the EGFRvIII mutation, as depicted in fig. 1, Ct (actb) 17.4, Ct (EGFRvIII) 20.1, Δ Ct 2.7, so the sample of fig. 1 is positive for the EGFRvIII mutation.
The immunohistochemical test is carried out on the sample 1, the test result is shown in figure 3, as can be seen from figure 3, the tumor cells of the sample 1 show membrane positivity, are EGFRvIII positivity, and are matched with the quick detection result, and the quick detection result has credibility and accuracy.
[ example 2 ]
A method for rapidly detecting EGFRvIII mutation comprises the following steps:
(1) extracting RNA of tumor tissue of the sample 1;
(2) constructing a PCR reaction system;
(3) performing real-time fluorescent quantitative PCR by a TaqMan probe method;
(4) and (5) judging the result.
Further, the extraction procedure for sample 1RNA was as follows: dewaxing a paraffin sample: 5 paraffin tissue sections of 7 μm samples 1 were placed in a 1.5ml centrifuge tube, 1ml xylene was added, centrifugation was carried out at 13,000Xg for 1 minute, the supernatant was discarded, 1ml xylene was added, centrifugation was carried out at 13,000Xg for 1 minute, and the supernatant was discarded. Adding 1ml of absolute ethyl alcohol into a centrifuge tube, centrifuging for 1 minute at 13,000Xg, removing supernatant, and airing; adding tissue lysate and proteinase K into the air-dried tissue, splitting at 56 ℃ for 15 minutes, transferring the centrifuge tube to 80 ℃, keeping for 15 minutes, taking down, adding DNase into the centrifuge tube after the centrifuge tube is moved to room temperature, and standing for 15 minutes at room temperature; adding a binding buffer solution and absolute ethyl alcohol into a sample, gently mixing uniformly, transferring the sample to an RNA extraction filter column, centrifuging for 1 minute at 13,000Xg, adding 600 mu L of a washing buffer solution into the sample, centrifuging for 1 minute at 13,000Xg, and repeatedly washing for three times; putting the filter column into a new 1.5ml centrifuge tube, placing the centrifuge tube at 56 ℃, opening the cover, airing for 3 minutes, adding eluent, covering the cover, and keeping at 56 ℃ for 2 minutes; the sample 1RNA sample was obtained by centrifugation at 13,000Xg for 1 minute.
Further, the PCR reaction system is constructed as follows: firstly, 2 mu L of RNA is taken as a template for constructing cDNA, and a housekeeping gene ACTB is introduced into a reaction system, so that the existence of negative control is ensured, the risk of false positive is reduced, the result interpretation is facilitated, and the specific reaction system comprises the following components:
| 20 μ L reverse transcription system component | Volume (μ L) |
| 2×One Step RT-PCR BufferⅢ(Takara) | 10 |
| TaKaRa Ex Taq HS(5U/μL)(Takara) | 0.4 |
| PrimeScript RT Enzyme MixⅡ(Takara) | 0.4 |
| EGFRvIII upstream primer (10. mu.M) | 0.4 |
| EGFRvIII downstream primer (10. mu.M) | 0.4 |
| EGFRvIII TaqMan probe | 0.8 |
| ACTB upstream primer (10. mu.M) | 0.4 |
| ACTB downstream primer (10. mu.M) | 0.4 |
| ACTB TaqMan probes | 0.8 |
| |
2 |
| RNase Free dH2O | 4 |
| |
20 |
The three processes of cDNA construction, PCR product amplification and fluorescent signal collection are realized by changing the reaction temperature of a reaction system, and the specific reaction program is as follows:
and after the reaction system is constructed, performing TaqMan real-time PCR to detect the EGFRvIII mutation condition.
Further, the step (4) is specifically as follows: determining whether the sample is positive for the EGFRvIII mutation or not according to the numerical size of the delta Ct, wherein the delta Ct is Ct (EGFRvIII) -Ct (ACTB); if delta Ct is more than 0 and less than 10, the sample is positive for EGFRvIII mutation; increasing the dosage of RNA and detecting again, wherein the dosage of the delta Ct is 10; and the delta Ct is more than 10 or no EGFRvIII specific fluorescent new number is collected, and the sample is EGFRvIII negative.
Further, the sequence of the EGFRvIII upstream primer is 5 'AGTCGGGCTCTGGAGGAAA-3', the sequence of the EGFRvIII downstream primer is 5 'TCCATCTCATAGCTGTCGGC-3', and the sequence of the EGFRvIII TaqMan probe is 5 '-FAM-AGAAAGGTAATTAT-MGB-3'; the sequence of the ACTB upstream primer is 5 'CTTCGCGGGCGACGAT-3', the sequence of the ACTB downstream primer is 5 'ATAGGAATCCTTCTGACCCATGC-3', and the sequence of the ACTB TaqMan probe is 5 '-HEX-CGGGCCGTCTTCCC-MGB-3'.
The amplification map obtained by TaqMan real-time PCR of sample 2 in this example is shown in FIG. 2: ● in fig. 2 represents the HEX fluorescence signal, negative control ACTB, and no FAM signal was collected, i.e., no amplification of the EGFRvIII mutation, so this sample was a wild sample.
The PCR products obtained from sample 1 and sample 2 in the examples were electrophoresed for 15 minutes in a 1 XTAE buffer under a 3% agarose gel at 180V to obtain an electrophoretogram as shown in FIG. 4. As seen from FIG. 4, the EGFRvIII primers designed by the present invention can clearly distinguish the EGFRvIII mutation from the EGFR wild type.
The PCR products obtained from sample 1 in the example and sample 2 in the example were sequenced, and the sequencing results are shown in fig. 5, the line frame is marked by the break point of exon 1 of EGFR linked to exon 8, and the sequence was reacted to form glycine inserted at the break point of the amino acid sequence, which is consistent with the expectation.
Through electrophoresis experiments and test results, the amplification product of the primer designed by the invention is EGFRvIII.
The above embodiments are only exemplary embodiments of the present invention, and are not intended to limit the present invention, and the scope of the present invention is defined by the claims. Various modifications and equivalents may be made by those skilled in the art within the spirit and scope of the present invention, and such modifications and equivalents should also be considered as falling within the scope of the present invention.
Claims (4)
1. A method for rapidly detecting EGFRvIII mutation is characterized by comprising the following steps:
(1) extracting RNA of the tumor tissue;
(2) constructing a PCR reaction system;
(3) performing real-time fluorescent quantitative PCR by a TaqMan probe method;
(4) and (5) judging the result.
2. The method for rapidly detecting the EGFRvIII mutation according to claim 1, wherein the step (2) is specifically as follows: adding reverse transcriptase, polymerase, EGFRvIII upstream and downstream primers, an EGFRvIII TaqMan probe, an ACTB upstream and downstream primer and an ACTB TaqMan probe into the same reaction system by taking the RNA extracted in the step (1) as a template; the three processes of cDNA construction, PCR product amplification and fluorescent signal collection are realized by changing the reaction temperature of a PCR reaction system.
3. The method for rapidly detecting the EGFRvIII mutation according to claim 1, wherein the step (4) is specifically as follows: determining whether the sample is positive for the EGFRvIII mutation or not according to the numerical size of the delta Ct, wherein the delta Ct is Ct (EGFRvIII) -Ct (ACTB); if delta Ct is more than 0 and less than 10, the sample is positive for EGFRvIII mutation; increasing the dosage of RNA and detecting again, wherein the dosage of the delta Ct is 10; and the delta Ct is more than 10 or no EGFRvIII specific fluorescent new number is collected, and the sample is EGFRvIII negative.
4. The method for rapidly detecting the EGFRvIII mutation is characterized in that the sequence of the EGFRvIII upstream primer is 5 'AGTCGGGCTCTGGAGGAAA-3', the sequence of the EGFRvIII downstream primer is 5 'TCCATCTCATAGCTGTCGGC-3', and the sequence of the EGFRvIII TaqMan probe is 5 '-FAM-AGAAAGGTAATTAT-MGB-3'; the sequence of the ACTB upstream primer is 5 'CTTCGCGGGCGACGAT-3', the sequence of the ACTB downstream primer is 5 'ATAGGAATCCTTCTGACCCATGC-3', and the sequence of the ACTB TaqMan probe is 5 '-HEX-CGGGCCGTCTTCCC-MGB-3'.
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Citations (3)
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|---|---|---|---|---|
| CN101130818A (en) * | 2007-07-03 | 2008-02-27 | 浙江大学 | Reagent kit for detecting epidermal growth factor acceptor third type mutant by real-time fluorescence quantitative PCR |
| CN110863053A (en) * | 2019-12-18 | 2020-03-06 | 广州迈景基因医学科技有限公司 | Primer, probe and method for detecting EGFR vIII mutant |
| CN110964829A (en) * | 2019-12-30 | 2020-04-07 | 江苏中方基因生物医学科技有限公司 | Application method of human RCN3-SSU72 gene fusion mutation detection primer combination, probe and detection kit |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101130818A (en) * | 2007-07-03 | 2008-02-27 | 浙江大学 | Reagent kit for detecting epidermal growth factor acceptor third type mutant by real-time fluorescence quantitative PCR |
| CN110863053A (en) * | 2019-12-18 | 2020-03-06 | 广州迈景基因医学科技有限公司 | Primer, probe and method for detecting EGFR vIII mutant |
| CN110964829A (en) * | 2019-12-30 | 2020-04-07 | 江苏中方基因生物医学科技有限公司 | Application method of human RCN3-SSU72 gene fusion mutation detection primer combination, probe and detection kit |
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| BARBARA OLDRINI ET AL.: "GFR feedback-inhibition by Ran-binding protein 6 is disrupted in cancer" * |
| GENBANK: AY588246.1: "Homo sapiens epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian) (EGFR) gene, complete cds" * |
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